Specificity in Endothelial Cell Calcium Signaling. The endothelial receives and integrates a variety of stimuli related to blood flow control (i.e. shear stress and vasoactive mediators), inflammation, and growth/repair. Inappropriate or defective endothelial cell responses to these stimuli can lead to a variety of vascular pathologies. Transduction of signals from all of these stimuli relies, in part, on mobilization of intracellular calcium. In large vessel endothelial cells, intracellular calcium pools are managed by two sarco (endothelial cell) plasmic calcium ATPases (SERCA); the uniquely expressed SERCA3 and the ubiquitous SERCA2b pumps. Mice lacking SERCA3 (SERCA3 knockout) are born viable with intact vasculature. However, aortas from these mice fail to respond to the vasodilator acetylcholine. Furthermore, SERCA3-deficient aortic endothelial cells do not generate a calcium transient in response to acetylcholine. The defects in endothelial cell activity present in the SERCA3 knockout mouse occur even though SERCA2b is present. These observations have lead to the hypothesis that endothelial cells utilize distinct intracellular calcium pools, as defined by these two SERCA pumps, to mediate responses to different types of stimuli. The goal of this project is to test this hypothesis, through completion of four specific aims, by demonstrating that the calcium pool maintained by SERCA3 participates in a subset of endothelial cell responses and that this calcium pool is segregated from the SERCA2b-managed pool within the endothelial cell. Specific aims 1 and 2 will characterize the distribution and the spatial organization of the SERCA3- and SERCA2b- managed calcium in the generation of calcium transients and endothelial cell responses induced by four classes of vascular stimuli (shear stress, vasoactivity, inflammation, and vascular repair) in endothelial cells from these mice will provide the experimental basis for these studies. Selective differences observed between cells and vessels of the two mice can be attributed to the absence of calcium stores established by SERCA3 and indicate those processes relying on this calcium pool. Completion of these aims will provide further insight into how specificity in calcium-mediated endothelial cell signal transduction is established and thus provide possible avenues for specific therapies directed at treating vascular disease.